The present invention relates to a method of treatment to achieve neuroprotection of cells in the central nervous system from death and for stimulating nerve cell survival in subjects with neurodegenerative disorders.
Pathological conditions resulting from the accelerated or ongoing death of nerve cells in the central nervous system are prevalent in today""s society and include acute or chronic neurodegenerative disorders. An example of a chronic neurodegenerative disorder is Alzheimer""s disease. Other examples of neurodegenerative disorders include Parkinson""s disease; Huntington""s disease; AIDS Dementia; Wernicke-Korsakoff""s related dementia (alcohol induced dementia); age related dementia; age associated memory impairment; brain cell loss due to head trauma, stroke, hypoglycemia, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma or epilepsy; spinal cord cell loss due to any of the conditions listed under brain cell loss; and peripheral neuropathy. Chronic and acute neurodegenerative diseases and acute nerve cell injury, as well as associated mortality and morbidity, have been largely untreatable with previous methods. Patient disability resulting from these conditions can cause a significant reduction in quality of life. In addition, these conditions impose a high cost to the patient and to society for long term care. Accordingly, effective therapeutic approaches directed to the prevention or reduction of nerve cell death or nerve cell damage associated with neurodegenerative diseases and acute nerve cell injury are needed. Specifically, an efficacious method for treating conditions in the brain resulting from neuron loss is needed that is relatively non-toxic, suitable for use in both females and males, and which can readily access the brain across the blood-brain barrier. The terms xe2x80x9cnerve cellsxe2x80x9d and xe2x80x9cneuronsxe2x80x9d are used interchangeably herein to refer to cells in the central nervous system, including the brain.
The amino acids glutamate and aspartate are known to function as excitatory neurotransmitters in the mammalian central nervous system. These amino acids activate a large series of excitatory neurotransmitter receptors known as xe2x80x9cglutamate receptorsxe2x80x9d. These glutamate receptors, along with the various neurotransmitters which activate them, make up the xe2x80x9cglutamatergic systemxe2x80x9d, the dominant excitatory nerve impulse transmission system of the mammalian central nervous system. A detailed explanation of the functioning of the glutamatergic system can be found in Olney, et al., U.S. Pat. No. 5,902,815.
Over the last few years, it has come to be recognized that overstimulation of excitatory neurotransmitter receptors can have serious pathological consequences for nerve cells. The overstimulation of cultured nerve cells in vitro by glutamate, for example, can lead to nerve cell death. Nerve cell death caused by such overstimulation is referred to as xe2x80x9cexcitotoxicity.xe2x80x9d Excitotoxicity is thought to be important in the pathogenesis of several neurodegenerative disorders, including stroke and ischemic injury. Excitotoxicity has been studied in vivo and in vitro, including in organotypic hippocampal explant preparations. Excitotoxicity is caused by Ca2+ overload resulting from its influx through highly Ca2+-permeable glutamate receptors sensitive to N-methyl-D-aspartate (xe2x80x9cNMDAxe2x80x9d) receptors and the resulting activation of intracellular kinases. One of the first events associated with excitotoxicity and nerve cell death is activation of protein kinase C (xe2x80x9cPKCxe2x80x9d). It has been shown that inhibition or reduction of PKC formation protects against nerve cell death following ischemia.
PKC refers to a family of more than ten Ca2+/phospholipid-dependent and independent threonine-serine kinase isozymes which regulate a multitude of mechanisms including cell differentiation and response to injury. PKC is abundant in neurons. It has been established that ischemia affects PKC activity and distribution. Ischemic nerve cell death has been associated with induction of PKC-delta isozyme. This effect can be blocked by NMDA inhibitors. Increased PKC-gamma immunoreactivity following incomplete ischemia has been found in the hippocampus. It has been shown that NMDA receptor stimulation can trigger PKC-gamma and beta isozyme activation. Protection of cultures by the NMDA antagonist MK-801 (dizocilpine maleate) has been associated with increases in PKC gamma, lambda, iota and micron isozymes, and PKC epsilon. PKC alpha, beta1, beta2 remained unchanged suggesting that PKC isozymes can play a significant role in regulating cell death. These data are consistent with the view that inhibition of PKC can confer neuroprotection.
Several PKC isozymes (for example, PKC-delta and epsilon) activate the mitogen-activated protein kinase (MAPK) cascade. The MAPK family consists of key regulatory proteins that are known to regulate cellular responses to both proliferative and stress signals. MAPK is abundantly expressed in nerve cells and may be necessary for cellular commitment to apoptosis. Apoptosis, also know as xe2x80x9cprogrammed cell deathxe2x80x9d, is a mechanism of nerve cell death initiated by activation of intracellular enzymes known as caspases. When a cell is undergoing the apoptotic process, its membrane disintegrates which exposes the inside of the membrane""s lipid bilayer. This process is sometimes referred to as the xe2x80x9cphosphatidylserine flip-flop.xe2x80x9d
It is believed that nerve cells will enter apoptosis if they are stimulated with a mitogenic agent, forcing them to divide. Nerve cells normally cannot divide, and if forced to divide, they instead enter the apoptosis program and die. Nerve cells may enter the division cycle when the MAPK cascade is activated. Accordingly, blocking the MAPK cascade can protect nerve cells from death.
MAPKs consist of several enzymes, including a subfamily of extracellular signal-activated kinases (ERK1 and ERK2) and stress-activated MAPKs. There are three distinct groups of MAPKs in mammalian cells: a) extracellular signal-regulated kinases (ERKs), b) c-Jun N-terminal kinases (JNKs) and c) stress activated protein kinases (SAPKs). As used herein, the term xe2x80x9cMAPK cascadexe2x80x9d refers to those protein kinases or protein kinase cascades located within nerve cells that are inhibited by 2-(2xe2x80x2amino-3xe2x80x2 methoxphenyll)-oxanaphthalen-4-one(referred to hereinxe2x80x9d PD098059), apigenin, or other similar bioflavonoids and which, upon activation, activate various transcription factors. Other as yet uncharacterized protein kinases which are similarly inhibited by PD098059 or apigenin and which are located within nerve cells, and which, upon activation, activate various transcription factors, are also within the scope of the term xe2x80x9cMAPK cascadexe2x80x9d.
An example of the MAPK cascade can be described as follows. PKC activation or other factors (e.g. increases in free intracellular Ca2+) activates small proteins called Ras/Raf-1, which in turn activate MAPK/ERK kinases referred to as MEKs. The MEKS in turn activate ERKs. The ERKS translocate to the cell nucleus where they activate transcription factors and thereby regulate cell proliferation. The inhibition of these protein kinases produces neuroprotective and neuron-treating effects as does the inhibition of the MAPK cascade. Examples of such kinases are mitogen-activated protein kinase 1 and 2, their homologues and isoforms, extracellular signal-regulated kinases (ERKs) their homologues and isoforms (ERK1, ERK2, ERK3, ERK4), and a group of kinases known as MAP/ERK kinases 1 and 2 or MEK1/2.
Exposure of cells to stress activates protein kinases by a variety of mechanisms. For example, ischemia, NMDA and amyloid peptides activate MAPK. Studies of functional roles of MAPKs in nerve tissue suggest that MAPK could be an important regulator of nerve cell death and plasticity. Thus, MAPK activation is required for hippocampal long-term potentiation (LTP). Okadaic acid, an inhibitor of protein phosphatase 2A, which dephosphorylates MEK1/2 and thus enhances and prolongs its activity, increased nerve cell death in cultured hippocampal slices. The experiments described in this application revealed that substances which are inhibitors of the MAPK cascade are neuroprotective.
Alzheimer""s Disease (AD) is a progressive neurodegenerative disease which is histologically characterized by an accumulation of neuritic plaques and neurofibrillary tangles and by neuron death. A major component of these neuritic plaques is the xcex2-protein, which is derived from a precursor protein called the xcex2-amyloid precursor protein (APP). The xcex2-amyloid protein itself has been shown to be toxic to neurons in vitro.
The xcex2-amyloid protein has, moreover, also been shown to potentiate the excitotoxicity caused by the overstimulation of glutamate receptors. When mature neuron cultures are exposed to doses of xcex2-amyloid that are too low to cause neuron degeneration by themselves and are then subsequently exposed to sublethal doses of glutamate, massive neuron death occurs. In addition to potentiating the toxicity of glutamate, xcex2-amyloid has been shown to increase the toxicity to neurons of exposure to NMDA and kainate, which are agonists of specific glutamate receptors. There is also evidence that xcex2-amyloid exposure specifically makes neurons more susceptible to injury or death. The present invention provides a method to block this effect of xcex2-amyloid by administering MAPK cascade inhibitors, thereby protecting neurons from death.
As described in detail herein, experiments demonstrating the neuroprotective properties of the bioflavonoids have been conducted using propidium iodide assays. Propidium iodide is a nuclear stain that binds RNA or DNA with little or no base pair preference. It can be excited by argon laser (488-547 nm), and the resulting emission can be collected in the red wavelengths of the spectrum. Propidium iodide is excluded from neurons that retain membrane integrity which makes it a useful and widely used marker of cell death. Recent studies show that, in severely apoptotic neurons, membrane permeabilisation occurs and they become stained by propidium iodide. This is the same stage of apoptosis when DNA laddering and phosphatidylserine flip-flops occur, and activity of caspases is high. Propidium iodide does not distinguish between apoptosis and necrosis.
As described above, at least two intracellular kinase systems may be involved in regulation of nerve cell death regardless of the initial mechanism of their activation. Therapeutic agents that are selected on the basis of their capability to inhibit these kinase systems may have generalized utility in treating nerve cell loss caused by disease or trauma in patients. Accordingly, it is an object of the present invention to provide a method for administering a class of compounds that have demonstrated biological efficacy in protecting neurons from cell death, where such compounds could be used in the treatment of the chronic as well as the acute conditions caused by neurodegenerative diseases, trauma, and aging at non-toxic dosages.
The present invention provides a method for protecting nerve cells from deterioration and cell death arising from disease, trauma or aging and may be used to achieve a similar effect in male and female subjects with minimal adverse side effects.
One aspect of the present invention involves a method of treating nervous system cells by administering a therapeutically effective dose of a MAPK cascade antagonist which is effective in preventing or treating damage to nerve cells caused by exposure of the cells to NMDA. The cells can be mammalian nervous system cells, and the cells can be treated in vivo. Alternatively, the nervous system cells can be treated in vitro. MAPK cascade antagonists useful in treating cells of the nervous system include, for example, PD098059 and apigenin, or pharmaceutically acceptable salts of these compounds.
Another aspect of the present invention involves a method of treating nervous system cells by administering a therapeutically effective dose of a MAPK cascade antagonist which is effective in preventing or treating damage to the nerve cells caused by the withdrawal of growth factors from the culture medium. The cells can be mammalian nervous system cells, and the cells can be treated in vivo. Alternatively, the nervous system cells can be treated in vitro. MAPK cascade antagonists useful in treating cells of the nervous system include, for example, PD098059 and apigenin, or pharmaceutically acceptable salts of these compounds.